B cells play a critical role in the pathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). A major focus of Project I is to define the bone marrow developmental cues and peripheral turnover of transitional B cells and how cytokine milieu, which may be perturbed in systemic autoimmunity, regulates this process. It is hypothesized that autoimmune disease is as.sociated with dysregulation of transitional B cell homeostasis during B cell development with alterations in the numbers of transitional B cells emerging from the bone marrow (BM), the inicroenvironmenlal signals that modulate this process (IFN, TNF), and the signaling threshold for progression to the mature compartinent (BAFF). On the other hand, we have found that SLE patients with a prominent expansion of circulating transitional cells after B cell depletion therapy (BCDT) enter long-term clinical remission, whereas both SLE and RA patients with more rapid memory reconstitution experience earlier relapse of disease. The focus of this proposal is to understand early B cell homeostasis and the factors that regulate B cell reconstitution after BCDT in SLE and RA through the following specific aims which will define: 1. the perturbations in homeostasis of human transitional B cells in autoimmune di.sease;2. the factors which regulate the balance of reconstitution of distinct B cell subsets after BCDT;and 3. the reciprocal regulation of transitional B cells and regulatory T cells and how this is altered in autoimmune disease and after BCDT. Specifically, we will define the dynamics of B cell development in humans using B cell depletion as a tool and multi-parameter flow cytometry. The lifespan and turnover of transitional B cells will be assessed by heavy water labeling, replication history, cell cycle, and delineation of survival and .selection. The effects of cytokine milieu on new BM B cell lyinphopoeisis will be examined. This project will explore the hypothesis that the outcome of BCDT rellects the balance between protective (regulatory, anti-inflammatory) and effector (prointlammatoiy) B cells and their corresponding cytokines. Transitional B cell functions to delineate include the production of anti-intlammatory cytokines such as IL-10 and TGFB and support of regulatory T cell (Treg) development. We will define whether human B cell subsets differentially support Treg expansion or are differentially susceptible to Treg suppression, if this function changes in autoimmune settings, and how these abnormalities are modified by BCDT. Elucidation of the homeostatic regulation of human transitional B cells will represent a major advance in human B cell biology. The research proposed will also help us understand how B cell development is dysregulated in autoimmune disease and how depletion induces improvement and, in some cases, long-lasting disea.se remission.